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Objective: HIV-associated nephropathy (HIVAN) is the most common cause of end-stage renal disease in persons with HIV/AIDS and is characterized by focal glomerulosclerosis and dysregulated renal tubular epithelial cell (RTEC) proliferation and apoptosis. HIV-1 viral protein r (Vpr) has been implicated in HIV-induced RTEC apoptosis but the mechanisms of Vpr-induced RTEC apoptosis are unknown. The aim of this study was therefore to determine the mechanisms of Vpr-induced apoptosis in RTEC.

Methods: Apoptosis and caspase activation were analyzed in human RTEC (HK2) after transduction with Vpr-expressing and control lentiviral vectors. Bax and BID were inhibited with lentiviral shRNA, and ERK activation was blocked with the MEK1,2 inhibitor, U0126.

Introduction

HIV-associated nephropathy (HIVAN) is the most common cause of end-stage renal disease in HIV-seropositive patients and results from direct infection of renal epithelial cells [1,2]. In addition to its role in glomerular pathogenesis [3,4], recent studies have demonstrated that HIV-1 viral protein r (Vpr) induces dysregulation of cytokinesis and apoptosis in renal tubular epithelial cells (RTECs) in vitro, which correlate with RTEC hypertrophy and apoptosis in HIVAN biopsy specimens [5].

Vpr induces apoptosis in many cell types; however, the mechanisms by which it induces cell death vary and are often cell-type-specific (reviewed in [6]), with both caspase-8 and caspase-9-mediated mechanisms having been implicated [7–9]. In some nonrenal cell types, Vpr induces activation of cellular DNA damage responses leading to G2/M arrest and Bcl-2–associated X protein (Bax)-dependent apoptosis[10–14]; in others, Vpr directly injures mitochondria [15].

Mitogen-activated protein kinases (MAPKs) are important mediators of HIV-induced renal pathogenesis [15,16]. Whereas Extracellular signal-regulated kinase (ERK) activation is classically associated with promoting cellular proliferation [17], it can also induce cell cycle arrest and/or apoptosis, particularly in the presence of cellular stressors such as DNA damage [18,19]. Sustained activation of ERK can induce apoptosis in neurons via a caspase-8-dependent pathway that is independent of Fas or FAS-associating death domain (FADD) [18,19].

Here, we report that transduction of a human RTEC line (HK2) with Vpr induced mitochondrial damage and apoptosis that was dependent upon activation of caspase-8 and caspase-9. Knock-down of BID and/or Bax expression using lentiviral shRNA vectors suppressed Vpr-induced apoptosis. We then demonstrate that Vpr-induced apoptosis was associated with prolonged ERK activation, and that inhibition of ERK activation with the specific MEK inhibitor U0126 reduced Vpr-induced apoptosis, caspase-8 and caspase-9 activation, and BID cleavage to tBID. Importantly, activated ERK was detected in RTEC in HIVAN patient biopsy specimens, strongly suggesting that these findings are relevant to HIVAN pathogenesis in vivo.

For mitochondrial assays using JC-1, we used VSV-pseudotyped lentiviral vectors encoding hexahistidine (His) and hemagglutinin (HA)-tagged Vpr (pHR-His-HA-VPR-IRES-GFP, abbreviated pHA-Vpr) and His-HA-tagged pHA-Vpr (Q65R) (inactive mutant) in which the start codon for GFP was mutated and a premature stop codon was added to GFP using the QuickChange site directed mutagenesis kit (Stratagene). pHA-Vpr and pHA-Vpr (Q65R) were gifts of Vincente Planelles, University of Utah [22].

Cell cycle analysis

Cells were collected by trypsinization, fixed in 50% ethanol, stained with propidium iodide (Invitrogen) and treated with 0.01 μg/μl percentage RNAse A (Puregement, Gentra Systems). Cell cycle parameters were measured using the FACScaliber flow cytometer equipped with CellQuest Software in the Mount Sinai Flow Cytometry Shared Research Facility and analyzed using FlowJo software version 6.4.7. Cell fragments were excluded from analysis on the forward scatter/side scatter plot. Gates from control transductions were applied to other experimental groups. Each experiment was conducted at least three separate times and representative plots are provided.

Real-time PCR

RNA was extracted using the RNeasy Mini Kit (Qiagen). cDNA was made using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). qPCR was performed using QuantiTect SYBR Green PCR Kit (Qiagen) at the Mount Sinai Quantitative PCR Shared Research Facility according to the protocol: 95C 15 min, 40 cycles of 94C 15 s, 58C 30 s, 72C 30 s. Primers were to Bax (sense: 5′-TTTGCTTCAGGGTTTCATCC, antisense: 5′-CAGTTGAAGTTGCCGTCAGA) with cyclophilin as a housekeeping gene (sense: 5′-AGGGTGGTGACTTTACACGC, antisense: 5′-ATCCAGCCATTCAGTCTTGG). The threshold cycle (Ct) for cyclophilin was subtracted from the Ct for each transcript (ΔCt). The fold-change difference in expression for each gene was defined as 2(ΔCt experiment - ΔCt control). Unpaired t-tests with a two-sided P value of less than 0.05 were used to compare the expression of each gene.

Mitochondrial assays

To assess the integrity of mitochondrial membrane polarization (MMP), cells were incubated with Mitotracker RedCMXRos (Invitrogen) for 30 min, trypsinized and analyzed by flow cytometry. MMP was also assessed by incubating cells with 5 μg/ml of JC-1 (Anaspec) prior to flow cytometric analysis of red and green fluorescence.

Caspase activation assays

Activation of Caspase 3/7, 8 and 9 was measured using Caspase Glo Assay kits (Promega). Luciferase activity was measured using a Wallac 1420 Manager Plate Read (Perkin Elmer). CaspaseGlo data are represented as the average of five experiments conducted in parallel.

Renal biopsy specimens and immunohistochemistry

Human biopsy material was collected previously from HIV-infected patients after informed consent under a protocol approved by the Mount Sinai Institutional Review Board. Kidney tissue was obtained from eight patients with HIVAN. Kidney biopsies from six HIV-seronegative patients with minimal change disease (MCD) were collected at Columbia University under a protocol approved by its Institutional Review Board. Immunostaining of paraffin-embedded renal biopsy specimens for phospho-ERK1,2 was conducted as described previously [15].

Statistical analysis

Flow cytometry data were analyzed using a chi-square test. Statistical significance was defined as P < 0.05. qPCR and caspase activation data were analyzed using the Student's t-test for populations with unequal variance and statistical significance was defined as two-sided (P < 0.05).

To further characterize whether Vpr expression induces mitochondrial injury in HK2 cells, Vpr and control-transduced cells were assayed by flow cytometry 5 days post transduction after incubation with Mitotracker RedCMXRos, a fluorescent dye whose accumulation in mitochondria is dependent upon intact MMP. Control-transduced cells displayed a single peak, indicating that cells were homogenous with respect to their content of oxidizing mitochondria (Fig. 1d). However, the HR-Vpr-transduced cells exhibited two peaks, one consisting of HK2 cells with less Mitotracker accumulation than control and another population with levels of Mitotracker accumulation that were similar to or higher than control (Fig. 1d). When the same cells were subsequently stained with propidium iodide, the percentage of hypodiploid Vpr-transduced cells was similar to the percentage of cells with reduced Mitotracker RedCMXRos staining (Fig. 1d), suggesting that the cells with reduced content of normal mitochondria were apoptotic. Whereas these data do not explain why some Vpr-transduced cells had increased Mitotracker RedCMXRos staining, since Vpr is known to induce cellular hypertrophy in HK2 cells [5], it is likely that this population consists of cells with a higher number of mitochondria that have not yet undergone apoptosis.

To confirm whether reduced Mitotracker RedCMXRos staining indicated Vpr-induced loss of MMP, we transduced cells with lentiviral vectors expressing either wild-type Vpr (HA-Vpr-ΔGFP) or mutated Vpr [HA-Vpr(Q65R)-ΔGFP] which is inactive due to mutation of its DCAF1 binding site [22]. GFP was mutated in these vectors to allow use of JC-1, a mitochondrial dye which shifts from red to green fluorescence when there is loss of MMP. Flow cytometric analysis of JC-1-stained HK-2 cells 5 days after transduction revealed that HA-Vpr-ΔGFP induced loss of MMP in 33.3% of cells, whereas MMP was reduced in only 6% of cells transduced with HA-Vpr(Q65R)-ΔGFP (Fig. 1e).

Vpr induces caspase-8 and caspase-9 activity in human kidney cells

Vpr has been shown to induce apoptosis by intrinsic and extrinsic pathways [7]. We therefore studied activation of caspase-8 and caspase-9 in Vpr-transduced HK2 cells. Five days after transduction with HR-Vpr or HR control vector, we found that caspases-8 and caspase-9 were both activated by Vpr as compared to control (Fig. 2a and b).

To determine whether activation of caspase-8 and 9 contributed to Vpr-induced apoptosis, the caspases were inhibited using Z-IETD-FMK and Z-LEHD-FMK, respectively. Inhibition of caspase-8 reduced apoptosis from 35 to 15.5% in HR-Vpr-transduced cells (P < 0.0001). Inhibition of caspase-9 reduced Vpr-induced apoptosis from 35 to 19.2% (P < 0.0001). Co-incubation with caspase-8 and caspase-9 inhibitors decreased apoptosis from 35 to 12.2% (P < 0.0001, Fig. 2c). These results confirmed that caspase-8 and 9 activation are important in Vpr-induced apoptosis in HK2 cells; however, their effects were not additive. There was no evidence that apoptosis was preferentially prevented in hyperploid or G2/M phase cells. Caspase inhibition also reduced death in cells transduced with the HR control vector.

Bax and BID mediate Vpr-induced cell death

Bax mediates Vpr-induced apoptosis in HeLa cells [11]. To investigate the role of Bax in Vpr-induced apoptosis in HK2 cells, we knocked down Bax expression using a lentiviral shRNA vector (shBax) prior to transduction with HR-Vpr or HR control vector. Transduction of HK2 cells with shBax reduced Bax mRNA expression by 89% in control-transduced cells and 83% in HR-Vpr-transduced cells compared to control shRNA (shLuc) (Fig. 3a). shBax transduction decreased the percentage of cells undergoing Vpr-induced apoptosis from 49 to 11.7% 5 days after transduction (Fig. 3b, P < 0.0005). Compared to cells transduced with HR-Vpr and shLuc, in cells transduced with Vpr and shBax, the decrease in cell death was associated with a concomitant increase in cells in G0/G1 (20.3 to 37.6%; P < 0.0005) and G2/M (14.3 to 30.5%; P < 0.0005). Knock-down of Bax also reduced death in control-transduced cells with concomitant small increases in each phase of the cell cycle.

In caspase-8-mediated apoptosis, caspase-8 cleaves BID to tBID and tBID subsequently promotes the proapoptotic activity of Bax. We therefore studied whether Vpr induces BID cleavage in HK2 cells. Whole cell extracts from Vpr and control-transduced cells were collected 3 and 5 days post transduction and analyzed by Western blotting using anti-BID antibody. tBID became detectable at 3 days and levels increased at 5 days post transduction with Vpr (Fig. 3c).

To determine whether BID is necessary for Vpr-induced apoptosis, BID expression was knocked down using lentiviral shRNA (shBID). Transduction of HK2 cells with shBID reduced BID protein levels (Fig. 3d). HK2 cells were transduced with shBID 2 days before transduction with HR-Vpr. Five days after transduction with HR-Vpr cells were stained with propidium iodide and analyzed by flow cytometry. shBID decreased the percentage of cells that became hypoploid after Vpr transduction from 35.2 to 9.8% (P < 0.0005). This decrease in cell death was associated with a corresponding increase in hyperploid cells from 16.6 to 34% (Fig. 3e; P < 0.0005) compared to cells transduced with shLuc control shRNA vector. Together, these experiments implicate BID in Vpr-induced cell death in HK2 cells.

To determine whether the effects of BID and Bax are additive, we simultaneously transduced HK2 cells with shBID and shBax 3 days before transduction with HR-Vpr and analyzed propidium iodide-stained cells 5 days later. Co-transduction with shBID and shBax reduced the Vpr-induced hypodiploid fraction from 21.3 to 6.7% (P < 0.0005), and increased the hyperploid fraction from 14.4 to 27.6% (Fig. 3f; P < 0.0005). Transduction of HK2 cells with shBID, shBax, or both vectors suppressed Vpr-induced cell death to levels one-third those of HR-Vpr with shLuc control (Fig. 3g). There was no evidence that the effects of shBID and shBax on cell death were additive.

Caspase 8 activation by Vpr is due to prolonged ERK activation

Mitogen-activated protein kinase pathways have been implicated as mediators of HIV renal pathogenesis [15] and prolonged ERK activation can induce caspase-8-dependent apoptosis[18]. JNK has a role in Vpr-induced apoptosis in monocytic cells [16]. We therefore studied whether Vpr induces activation of JNK and/or ERK in HK2 cells. Western blotting of total cell lysate collected after transduction with HR-Vpr and HR-control revealed no evidence of JNK activation (Fig. 4a), but there was a striking increase in ERK phosphorylation 1 and 5 days after Vpr transduction relative to control (Fig. 4b).

To determine whether ERK activation was necessary for activation of caspase-8, MEK1 and MEK2 (activators of ERK) were inhibited using the specific inhibitor U0126. Inhibition of ERK phosphorylation was confirmed by Western blotting of protein lysate from HR-Vpr and HR-control transduced cells after treatment with U0126 (Fig. 4c). Incubation with U0126 for the third and fourth days or all 5 days after transduction with Vpr inhibited cleavage of BID to tBID (Fig. 4c). These data demonstrate that prolonged inhibition of ERK activation diminished BID cleavage in Vpr-transduced cells.

We then studied whether treating Vpr-transduced HK2 cells with U0126 could prevent Vpr-induced apoptosis. Cells were transduced with HR-Vpr and subsequently incubated with U0126 for either all 5 days prior to analysis or for the final 2 days prior to analysis. Flow cytometric analysis of propidium iodide-stained cells revealed that MEK inhibition with U0126 for the final 2 days prior to analysis reduced the hypoploid fraction in Vpr-transduced cells from 28.1 to 22.1% (Fig. 4c; P < 0.0005). Incubation of U0126 for all 5 days after HR-Vpr transduction further decreased accumulation of hypodiploid cells to 16.1% (Fig. 4d; P < 0.0005). MEK inhibition with U0126 also had pronounced effects upon cell cycle dynamics in Vpr-transduced cells. Incubation with U0126 for the final 2 days prior to analysis eliminated the greater than 8N peak [Fig. 4d(v)] and incubation with U0126 for all 5 days following Vpr transduction decreased the percentage of hyperploid cells from 38.2 to 12.8% [Fig. 4d(vi); P < 0.0005] and induced a shift of hyperploid cells to the G2/M peak. Interestingly, MEK inhibition with U0126 had no effect upon the percentage of G0/G1 cells, suggesting that U0126 did not exert its effects by preventing cells from transitioning from G0/G1 to S phase but prevented Vpr-transduced cells from progressing from G2 through mitosis.

Since we had found that MEK inhibition with U0126 decreased Vpr-induced cell death, we studied the effect of U0126 upon caspase-8 and caspase-9 activation 5 days after transduction with Vpr. Incubation with U0126 reduced Vpr-induced activation of caspases-8 and 9 to levels found in control-transduced cells (Fig. 4e and f; P < 0.0001). These results demonstrate that ERK activation is required for Vpr-induced caspase-dependent apoptosis in HK2 cells.

ERK is activated in RTEC in patients with HIVAN

To determine whether ERK is activated in RTEC in HIVAN specimens in vivo, we performed immunohistochemistry to detect phospho-ERK in HIVAN biopsy samples and biopsy samples from patients with minimal change disease (MCD), a form of proteinuric renal disease in which, in contrast to HIVAN, patients rarely develop tubulointerstitial injury or progressive renal failure. We detected phospho-ERK in the RTEC in HIVAN biopsies but not MCD (Fig. 5a). These results confirm that the ERK activation that we observed in Vpr-transduced HK2 cells in vitro recapitulates findings in patients with HIVAN in vivo.

Discussion

In developed countries, widespread availability of antiretroviral therapy has led to dramatic reductions in morbidity and mortality due to opportunistic infections. Renal disease has become the fourth leading diagnosis contributing to death among Americans with HIV/AIDS [23] and HIVAN is the leading cause of ESRD in HIV-seropositive patients [24]. The mechanisms by which expression of viral genes in RTEC leads to tubulointerstitial disease, characterized by dysregulated RTEC apoptosis and proliferation, remain poorly characterized. Previous studies have described HIV-1 Vpr as a principal HIV gene responsible for inducing RTEC hypertrophy and apoptosis[5]. Here, we define the mechanisms of Vpr-induced apoptosis in human RTEC.

Vpr can induce apoptosis in many cells types but the mechanisms by which it does so differ depending upon cell type and context [6]. Most previous research has focused upon the ability of Vpr to injure mitochondria either by direct binding to the adenine nucleotide transporter on the outer mitochondrial membrane, or via up-regulation of Bax with resultant loss of mitochondrial membrane polarization and release of proapoptotic factors, culminating in caspase-9 activation and apoptosis[25,26].

When we found that Vpr induced apoptosis in RTEC, we initially hypothesized that the mechanism of apoptosis in these cells was via Bax-dependent mitochondrial injury, leading to activation of caspase-9 as reported in HeLa cells by Andersen et al.[11]. As we predicted, Vpr induced caspase-9 activation and we also found that Bax expression was important for Vpr-induced apoptosis. To our surprise, however, we also found that Vpr activated caspase-8 and that inhibition of caspase-8 was as effective in preventing cell death as caspase-9 inhibition. Moreover, caspase-9 inhibition did not reduce cell death more than caspase-8 inhibition alone, suggesting that caspase-9 might be acting downstream of caspase-8. Whereas our data demonstrating a role for caspase-8 in Vpr-induced apoptosis were unexpected, Vpr has been reported to induce caspase-8-mediated apoptosis in human neurons and CD8 cells [27,28].

Since active caspase-8 can cleave BID to tBID, which can then facilitate Bax-induced mitochondrial injury, we tested whether suppression of BID expression could prevent Vpr-induced apoptosis. Transduction of HK2 cells with shRNA directed against BID prevented apoptosis as effectively as Bax shRNA, and co-suppression of Bax and BID did not further reduce apoptosis, suggesting that BID is upstream of Bax-induced apoptosis in Vpr-transduced HK2 cells. Activation of caspase-8 is classically associated with receptor-mediated (extrinsic) apoptosis[29,30]. In preliminary studies, we were unable to inhibit Vpr-induced apoptosis using blocking antibodies directed against Fas, the TNF-α receptor, or the TRAIL receptor (data not shown). Conaldi et al.[31] also reported that HIV-1-induced apoptosis in human RTEC is not prevented by Fas blocking antibodies.

Previous studies have described the role of the HIV-1 Nef protein in ERK activation in podocytes. This study is the first to focus on ERK activation in RTEC in HIVAN and is also the first to demonstrate a role for Vpr-induced activation of ERK as a stimulus for apoptosis. Whereas other studies have reported a role for JNK activation in Vpr-induced apoptosis in nonrenal cell types [16], we found no evidence of JNK activation in Vpr-transduced HK2 cells. As expression of HIV-1 Nef can also activate ERK signaling in podocytes, the combined effects of Nef and Vpr on ERK signaling in RTEC require future study.

Our studies demonstrate that ERK activation is necessary for caspase-8 activation in Vpr-transduced RTEC and that inhibition of ERK kinases MEK1 and MEK2 with the highly specific inhibitor U0126 decreases caspase-8 activation and cleavage of BID to tBID. We therefore propose a model in which prolonged ERK activation results in caspase-8 activation, leading to cleavage of BID to tBID, which facilitates Bax-induced mitochondrial permeablization, caspase-9 activation, and cell death. This model is depicted in Fig. 5b, and is similar to the pathway described for neuronal apoptosis in response to oxidative stress [32] and in HEK 293 cells subjected to prolonged ERK activation [18]. The mechanism by which Vpr induces ERK activation in RTEC is unknown.

Though ERK activation is commonly associated with promotion of cellular proliferation, ERK signaling can also induce cell cycle arrest in several cell types [33,34]. Furthermore, prolonged ERK activation facilitates glutamate and oxidative stress-induced toxicity in neuronal cell lines and primary culture [19], and apoptosis in 293T cells [18]. ERK has also been implicated in vivo in a neuronal ischemic injury model [35]. Importantly, ERK activation occurs in response to various DNA damage stimuli [36], and mediates cell-cycle arrest and apoptosis after certain forms of DNA damage, effects that were inhibited by U0126 [37]. Recent studies have demonstrated that strong ERK activation causes cell cycle arrest via phosphorylation of Cdc25A [38]. Interestingly, the phosphorylation of Cdc25A induced by ERK activation is similar to Cdc25A phosphorylation by Chk1, which occurs in response to activation of DNA damage response pathways. Since Vpr has been shown to induce ATR-dependent Chk1 activation in some cell types [39], further research is needed to determine whether Vpr-induces ATR/Chk1 dependent cell cycle arrest in human RTEC. Moreover, Vpr has been recently demonstrated to activate DNA damage response in RTEC as detected by accumulation of γ-H2AX foci [40]. Since some DNA damaging agents including cisplatin, can induce ERK-dependent RTEC apoptosisin vitro and in vivo[41], future studies should further characterize Vpr-induced DNA damage response in RTEC and the mechanism by which it may lead to ERK activation.

In conclusion, these studies have identified a novel mechanism for Vpr-induced apoptosis in human RTEC. We have demonstrated that Vpr induces ERK-dependent activation of caspase-8, leading to cleavage of BID to tBID, and ultimately, caspase-9 activation and apoptosis. Moreover, ERK is activated in RTEC in biopsy samples from patients with HIVAN, a disease characterized by RTEC injury and apoptosis, but not in minimal change disease, a renal disease characterized by proteinuria without tubular injury or progressive renal failure.

Acknowledgements

Alexandra Snyder conducted the experiments, and in conjunction with Michael J. Ross, wrote the text and created the figures. Jeremy S. Leventhal performed studies analyzing BID knock-down at the protein level. Paul Rosenstiel assisted in analysis of ERK activation and Kevin Barley performed studies of JNK activation. Zygimantas C. Alsauskas performed studies on effects of Vpr on MMP. Pengfei Gong, and Justin J.K. Chan assisted in cell culture and generation of lentiviral vectors. John C. He performed immunohistochemistry of patient biopsy specimens. Mary E. Klotman, Michael J. Ross* and Paul E. Klotman provided guidance, supervision, and funding for this project.

The work was generously supported by NIH grants R01DK078510 (Ross) and P01DK056492 (Klotman) and an HHMI Medical Student Research Fellowship.